Chronic changes in electrical excitability profoundly affect synaptic transmission throughout the lifetime of a neuron. Activity-dependent plasticity can be manifest as the up- or down regulation of subsequent synaptic signaling, and can occur through both presynaptic and postsynaptic targets. The objective of this application is to use sophisticated imaging techniques to examine the neuronal response to increased electrical activity, as mimicked by tonic depolarization, in hippocampal neurons. The rationale is that if we can understand the signaling changes in neurons that accompany tonic depolarization, then we should gain insight into the homeostatic mechanisms that are initiated by drugs of abuse, as well as by neuronal injury and disease. In preliminary patch-clamp electrophysiology studies, tonic depolarization elicited a homeostatic decrease in subsequent glutamate neurotransmission. In the proposed study, it is hypothesized that a presynaptic alteration of the synapic vesicle pool accounts for this decrease. A selective, homeostatic depression of glutamate release from excitatory neurons may be a model for the response of synapses to drugs of abuse, many of which act by altering overall activity levels for prolonged periods. In order to characterize the deficits in glutamate signaling fully, immunohistochemistry, electron microscopy, and vital fluorescent dye (FM1-43) experiments are proposed to supplement the previous electrophysiology data. The combination of imaging and electrophysiological methods will also be used to define the molecular mechanisms responsible for the depression of glutamate transmission after tonic depolarization. Training in these advanced imaging techniques will greatly enhance the career development of the applicant, allowing her to assess synaptic function from many perspectives. Importantly, the scientific and technical expertise gained in the course of these studies will allow the candidate to attain her long-term goal of establishing an independent research career in neuroscience.